Genetic characterization of Cryptosporidium spp. among patients with gastrointestinal complaints.

Aim: This study investigated subtypes of Cryptosporidium in patients with gastrointestinal complaints in Tehran, Iran. Background: Cryptosporidium, an intracellular protozean parasite, is among the major causative agents of gastroenteritis disorders in humans. It also causes water-borne and food-borne outbreaks of diarrheal diseases. Patients and methods: A total of 1685 fecal samples were collected from patients with gastrointestinal complaints who had been referred to clinical laboratories Tehran, Iran. The primary diagnosis was established by the detection of oocysts using the modified Ziehl-Neelsen staining method and following that, the positive microscopically samples were selected for sequence analysis of the partial 60 kDa glycoprotein (gp60) gene. Results: Out of 1685 collected samples, 7 (0.4 %) were positive for Cryptosporidium oocysts. Sequence analysis of gp60 gene in seven Cryptosporidium isolates revealed that two subtype families were identified, IIa and IId. Five (of 7) isolates belonged to the subtype family IIa and the remaining two isolates belonged to IId. Two sub-types were recognized within the subtype family II,a including IIaA16G2R1 (3/5), IIaA17G1R1 (2/5), while IIdA17G1d was the only subtype within IId subtype family. Conclusion: The predominance of zoonotic subtype families of C. parvum species (IIa, IId) in this study highlights the importance of zoonotic transmission of cryptosporidiosis in the country.


Introduction
Intestinal protozoan parasites are still major health problems in tropical and subtropical areas and are characteristically found among people with a low socio-economic grade and poor hygiene (1). Among these intestinal parasites, zoonotic, contaminated water, foodborne or airborne contact (5)(6)(7). Currently, the use of molecular approaches in genetic characterization of Cryptosporidium spp. at polymorphic loci has allowed a better understanding of the epidemiology of cryptosporidiosis (2)(3). Molecular biology has well-known as a powerful tool for categorizing Cryptosporidium and has discovered major variation within the genus (about 30 species). Two predominantly species that have been found in humans are C. parvum and C. hominis. However, other species such as C. meleagridis, C. muris, C. felis, C. canis, C. suis and C. andersoni have been rarely detected in feces of immunocompetent and immunocompromised individuals (8).
In the past two decades, reports of cryptosporidiosis in Iran have been achieved using microscopy (9)(10)(11)(12)(13). Recently, researchers have developed PCR-based techniques for detection and identification of Cryptosporidium spp. (14)(15)(16)(17). Fingerprint of C. parvum infection has a critical role in outbreak investigations. DNA sequencing of the Cryptosporidium 60-kDa glycoprotein (GP60) gene has revealed substantial genetic heterogeneity among C. hominis and C. parvum isolates. GP60 gene could be used as a marker to determine the different subtype families within both species, including: Ia, Ib, Id, Ie, If and Ig for C. hominis and IIa, IIb, IIc, IId, IIe, IIf , IIg, IIh, IIi, IIk, and IIl for C. parvum (18). Within each subtype group, there are several subtypes primarily based on the number of tri-nucleotide repeats coding for the amino acid serine (2,7,19). To our knowledge, there are several molecular epidemiological studies that have documented the distribution of subtypes of cryptosporidium ssp. in children with diarrhea (8,15), animals (20) and environments (the water that isolated from rivers) (21) in Iran.
In this study, we identified the genotypes of the Cryptosporidium isolates from patients with gastrointestinal complaints referred to clinical laboratories of Tehran using the polymerase chain reaction (PCR) amplification and sequencing analyses of the Gp 60 gene.

Sampling
A total of 1685 fecal samples were collected from patients with gastrointestinal complaints who had been referred to Medical Centers in Tehran, Iran. Cryptosporidium oocysts were identified in samples after concentration by formalin-ethyl-acetate sedimentation and staining with a modified Zeihl-Neelsen technique (22). The positive Cryptosporidium spp. isolates were preserved in 2.5% potassium dichromate and kept at 4°C until DNA extraction.

DNA extraction
Approximately 300 μl of fecal suspension was washed three times with distilled water to remove trace of dichoromate and then genomic DNA was extracted using DNAzol kit according to the manufacturer's instructions (Invitrogen, life technologies, Cat. No 10503-027, USA) with the addition of three freeze-thaw cycles (10 minutes) after resuspending samples in lysis buffer (to rupture the Cryptosporidium oocysts). The oocysts were frozen in the liquid nitrogen tank. Thawing was carried out at 90° C in the water bath.

DNA subtyping and sequence analysis
For subtyping C. parvum and C. hominis, a fragment of about 400 bp of the gp60 gene was amplified by nested PCR with the primers 5_-ATA GTC TCC GCT GTA TTC-3_ and 5_-GCA GAG GAA CCAGCA TC-3_ in primary PCR and 5_-TCC GCT GTA TTC TCA GCC-3_ and 5_-GAG ATA TAT CTT GGT GCG-3_ in secondary PCR, as described (Abe et al., 2006). PCR products were visualized by electrophoresis in 1.5% agarose gels stained with ethidium bromide.
The PCR-amplified products were subjected to direct sequencing using a BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and a Genetic Analyzer PrismTM 3130x1 (Applied Biosystems). The secondary PCR products were sequenced in both directions and, if variations were found, results were confirmed by sequencing of at least two independent PCR products. All sequences were edited manually and analyzed with reference sequences using the GenRunner software (v. 3.05).
Subtypes were recognized based on the number of trinucleotide repeats (TCA or TCG) coding for the amino acid serine (19).

Statistical analysis
The prevalence of Cryptosporidium infection and prevalence of C. parvum and C. andersoni in pre-weaned calves was compared with prevalence data for post-weaned calves. The Chisquare test for independence was used to analyze the data and differences were considered significant when P < 0.05. Statistical analysis was performed using SPSS (Ver. 12).

Results
Among the 1685 patients (66.3% male and 33.7% female) included in this study, microscopic examinations of the specimens revealed the presence of Cryptosporidium oocysts in 7 (0.4 %) of the samples. Clinical information about these samples is presented in table 1. Patient complaints included abdominal pain, flatulence, tenesmus, diarrhea and dysentery. All samples were successfully amplified using specific primers ( Figure 1) and PCR products of GP60 gene were purified and sequenced using a genetic analyzer machine. The sequences were determined and analyzed using the chromas program and aligned with each other and with previously reported sequences for identification of the alleles and subtypes. The result of this analysis showed that all isolates were C. parvum species.

Discussion
There are wide intraspecific variations in C.
All Cryptosporidium isolates from patients with gastrointestinal complaints were identified as C.
parvum species and none of them belonged to C. hominis. Also, two main subtype families (IIa and IId) were recognized. Regarding to other studies, C.
parvum is reported in most cryptosporidiosis cases in Iran (6,14,24), which highlights the significance of zoonotic transmission of cryptosporidiosis in the country (25)(26).
parvum and C. hominis were characterized in children and cattle by sequence analysis of the gp60 gene, which showed cattle and children were mainly infected by C. parvum IIa subtypes and C.
parvum IIa and IId subtypes, respectively (17). In some countries such as Spain, Egypt and China, IId subtypes are known to be more prevalent in sheep and goats (40)(41)(42). Also, IId subtypes are also common in children from Iran neighboring countries (43)(44)(45)(46). Sharbatkhori and her collogues, showed three haplotypes of IIa subtype family including IIaA16G2R1, IIaA17G1R1, IIaA22G3R1 and one haplotype of IId subtype family among diarrheic children from Gonbad Kavoos City (Golestan Province, Northern Iran) and suggested a zoonotic transmission of cryptosporidiosis in this area (8).
The majority of IIa and IId subtypes highlight the significance of zoonotic Cryptosporidium transmission in Iran. Thus, cattle could be a plausible source of human infection with C.
In another study, in 2011, high diversity of Cryptosporidium sub-genotypes was shown among Malaysian HIV infected individuals. The results obtained from this paper signified the possibility of zoonotic as well as anthroponotic transmissions of cryptosporidiosis in HIV infected individuals (47). In 2015, Mahmoudi, et al. determined the genotype and subtype distribution of Cryptosporidium spp. in river water samples in Iran. They showed that all C. parvum and C. hominis isolates belonged to the IId and Id subtype families, respectively and this source is a potential risk of waterborne cryptosporidiosis in humans and animals (21).
Vieira, et al. identified two subtype families (IIa and IId) from children, calves and eight pigs in Romania. They proposed, cattle might be the source of Cryptosporidium infections for humans and the transmission dynamics of C. parvum in Romania (48).
In their study, Wang, et al. suggest that, due to the higher nucleotide diversity of C. parvum IId GP60 sequences, more population genetic studies using high-resolution tools are needed to present a better explanation of the origin and dissemination of C. parvum in the world (23).
In 2010, sequence analysis of the GP60 locus identified three C. parvum and two C. hominis subtype families in Jordan. In this study several rare and novel subtypes were reported as well (45).
Preliminary molecular epidemiological studies have revealed some unique features of cryptosporidiosis transmission in humans in Iran and other Mideast countries. As the C. parvum subtype family IId was the dominant family causing cryptosporidiosis in humans in Iran, zoonotic transmission could possibly be involved. However, more extensive sampling of both humans and farm animals, especially sheep and goats, and collection of epidemiological data in case-control and longitudinal studies are needed for a better understanding of the sources of C.
parvum infections in humans in Iran and other Mideast countries.